Work from several laboratories over the past 10-15 years has established that the monomeric end-products of lysosomal digestion, e.g. amino acids, monosaccharides, nucleosides and (probably) lipid components traverse the lysosomal membrane to the cytoplasm through the agency of specific carriers rather than by simple diffusion. The importance of these carriers to animal metabolism is indicated by the existence of two human disorders, viz., cystinosis and sialic acid storage disease, which owe their origin to inherited defects in the lysosomal transport systems for cystine and sialic acid,, respectively. To date, none of the aforementioned lysosomal transport systems has yet been purified or characterized by direct chemical approaches, nor has any of these carriers been cloned by genetic linkage studies or other recombinant DNA techniques. Although direct purification of membrane transportors is hampered by the lack of a suitable assay system, such difficulty has been overcome in the past by the use of reconstituted lipid membrane systems or proteoliposomes, i.e., lipid vesicles incorporating the membrane proteins and which retain the original transport function. As a preliminary to employing this methodology to the purification of the lysosomal cystine carrier, we have prepared lysosomal membrane vesicles from rat liver and have investigated their characteristics with respect to cystine transport. Our studies have indicated that these vesicles possess cystine transport activity and may therefore serve as useful starting material in the preparation of functional proteoliposomes for purification of the cystine carrier.